There have been some attempts by scientists to increase expression levels of transgenes in transgenic animals and plants by the introduction of a bacteriophageT7 polymerase/T7 promoter (T7-pol/pT7) system thereinto. T7 RNA polymerase (T7-pol) is a single polypeptide of −98 kDa which specifically recognized the short viral promoter pT7 [Dunn J. J. et al., J. Mol. Biol. 166:477–535 (1983); Moffatt B. A. et al. J. Mol. Biol. 173:265–269 (1984)]. T7-pol does not require auxiliary proteins for transcription [Chamberlin M., and Ryan T. The Enzymes Ed. Boyer P. D. Academic Press N.Y 15:87–108 (1982)] and recognizes a single (albeit nor stringent) terminator.
It was suggested that any gene placed under the control of the T7 promoter will be specifically and strongly transcribed by a cloned T7 RNA polymerase, which was proven to be successful in bacteria [Tabor S., and Richardson C., Proc. Natl. Acad. Sci USA 82:1074–1078 (1985); Studier F. W., and Moffatt, B. A., J. Mol. Biol. 189:113–130 (1986)], and somewhat successful in animal cells [Fuerst T. R., et al. Proc. Natl. Acad. Sci. USA 83:8122–8126 (1986); Fuerest T. R., et al. Mol. Cell. Biol. 7:2538–2544 (1987)] Dunn J. J., et al. Gene 68:259–266 (1988); Lieber, A., et al. Nucl. Acids Res. 17:8485–8493 (1989); M ss, B., et al. Nature 348:91–92 (1990)].
The situation in plants is more vague. The expression of T7-pol in tobacco protoplasts and the NLS-direction of the expressed enzyme to the nucleus has been reported [Lassner M. W. et al. Plant Mol. Biol. 17:229–234 (1991)]. In addition, the expression of either T7-pol or a reporter gene driven by the T7-pol/pT7 system in plants was described [Caviedes, M. A., et al., Abstract #479 of the 4th Intl Congress of Plant Mol. Biol (1994); Tuttle A., et al. Abstract #478 of the 4th Intl Congress of Plant Mol. Biol (1994)]. A more successful T7-pol/pT7 expression system was described [Tuttle, A. et al. Abstracts of the 4th Int. Congress of Plant Mol Biol #478 (1994)] in which T7-pol expressed in tobacco but targeted to the protoplast, directed expression of GUS. A T7-derived system which promotes expression in plants was also described in the course of a novel method to produce hybrid seeds [U.S. Pat. No. 5,659,124] and in plastids which may be considered as prokaryotic cells [McBride, K. E. et al. Proc. Natl. Acad. Sci. USA 91:7301–7305 (1994); U.S. Pat. No. 5,545,817].
Gene silencing in transgenic plants is a documented phenomenon, which relates to the introduction of a foreign gene into a cell thereby inducing its silencing, rather than its expression [Cox K. H., and Goldberg R. B. in Plant Molecular Biology, A Practical Approach (Shaw, C. H., ed). Washing D.C. IRL Press, IRL Press, pp. 1–35 (1988); Baulcombe D.C., and English J. J., Curr. Opin. Biotechnol. 7:173–180 (1996); Meyer P., and Seedler, H., Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:23–48 (1996); Stam M., et al. Ann. Bot. 79:3–12 (1997)]. In general, the insertion of a particular gene into a plant may cause the silencing of homologous native or transgenic genes, which is referred to as “co-suppression” [Depicker A., et al. Curr. Opin. Cell Biol. 9:373–382 (1997); Matzke M. A., et al. EMBO J. 8:643–649 (1989); Matzke M. A., et al. Mol. Gen. Genet. 238:379–386 (1993); Napoli C., et al. Plant Cell 2:279–289 (1990)]. Co-suppression may also be caused by the introduction of homologous RNAs into the cells, such as viral RNAs [Lindbo J. A., et al. Plant Cell 5:1749–1759 (1993); Mueller E., et al. Plant J. 7:1001–1013 (1995)].
Silencing may occur at the transcriptional level, i.e. inhibition of transcription [Flavell R. B. Proc. Natl. Acad. Sci. USA 91:3490–3496 (1994); Matzke M. A. et al. in Homologous Recombination and Gene Silencing in Plants Ed. Paszkowski J. Kluwer Academic Publishers, Dordrecht 271–307] or post-transcriptionally [Van Blokland R. et al. Plant J. 6:861–877 (1994); B erjan W., et al. Plant Cell 6:1401–1414 (1994); De Carvalho-Niebel F., et al. Plant Cell 7:347–358 (1995)]. It has been suggested that silencing at the post-transcriptional level is caused by degradation of the primary transcript of the expressed gene, thus no mRNA and no protein will be found in the cytoplasm [Tanzer M. M., et al. Plant Cell 9:1411–1423 (1997)]. Furthermore, silencing-triggering mechanism involving the sensing of RNA levels [Goodwin J., et al. Plant Cell 8:95–105 (1996); Smith C. J. S., et al., Mol. Gen. Genet. 224:477–481 (1990); Metzlaff M., et al. Cell 88:845–854 (1997)] or ectopic pairing of homologous DNA sequences [Baulcombe D.C., and English J. J., Curr. Opin. Biotechnol. 7:173–180 (1996); Matzke M. A., Dev. Genet 11:214–223 (1990)] have also been suggested. Plant cells which have been silenced for a certain viral transgen and thus became resistant to virus infection due to the specific degradation of viral sequences have also been described [Lindbo J. A., et al. (1993) ibid.; Goodwin J., et al. (1996) ibid.].